Health related quality of life after combined hormone replacement therapy: randomised controlled trial.
To assess the effect of combined hormone replacement therapy (HRT) on health related quality of life. Randomised placebo controlled double blind trial. General practices in United Kingdom (384), Australia (94), and New Zealand (24). Postmenopausal women aged 50-69 at randomisation; 3721 women with a uterus were randomised to combined oestrogen and progestogen (n=1862) or placebo (n=1859). Data on health related quality of life at one year were available from 1043 and 1087 women, respectively. Conjugated equine oestrogen 0.625 mg plus medroxyprogesterone acetate 2.5/5.0 mg or matched placebo orally daily for one year. Health related quality of life and psychological wellbeing as measured by the women's health questionnaire. Changes in emotional and physical menopausal symptoms as measured by a symptoms questionnaire and depression by the Centre for Epidemiological Studies depression scale (CES-D). Overall health related quality of life and overall quality of life as measured by the European quality of life instrument (EuroQol) and visual analogue scale, respectively. After one year small but significant improvements were observed in three of nine components of the women's health questionnaire for those taking combined HRT compared with those taking placebo: vasomotor symptoms (P<0.001), sexual functioning (P<0.001), and sleep problems (P<0.001). Significantly fewer women in the combined HRT group reported hot flushes (P<0.001), night sweats (P<0.001), aching joints and muscles (P=0.001), insomnia (P<0.001), and vaginal dryness (P<0.001) than in the placebo group, but greater proportions reported breast tenderness (P<0.001) or vaginal discharge (P<0.001). Hot flushes were experienced in the combined HRT and placebo groups by 30% and 29% at trial entry and 9% and 25% at one year, respectively. No significant differences in other menopausal symptoms, depression, or overall quality of life were observed at one year. Combined HRT started many years after the menopause can improve health related quality of life. ISRCTN 63718836.


Process Development of Adenoviral Vector Production in Fixed Bed Bioreactor: From Bench to Commercial Scale.
Large-scale vector manufacturing for phase III and beyond has proven to be challenging. Upscaling the process with suspension cells is increasingly feasible, but many viral production applications are still applicable only in adherent settings. Scaling up the adherent system has proven to be troublesome. The iCELLis(®) disposable fixed-bed bioreactors offer a possible option for viral vector manufacturing in large quantities in an adherent environment. In this study, we have optimized adenovirus serotype 5 manufacturing using iCELLis Nano with a cultivation area up to 4 m(2). HEK293 cell cultivation, infection, and harvest of the virus (by lysing the cells inside the bioreactor) proved possible, reaching total yield of up to 1.6×10(14) viral particles (vp)/batch. The iCELLis 500 is designed to satisfy demand for large-scale requirements. Inoculating a large quantity of cell mass into the iCELLis 500 was achieved by first expanding the cell mass in suspension. Upscaling the process into an iCELLis 500/100 m(2) cultivation area cassette was practical and produced up to 6.1×10(15) vp. Flask productivity per cm(2) in iCELLis Nano and iCELLis 500 was in the same range. As a conclusion, we showed for the first time that iCELLis 500 equipment has provided an effective way to manufacture large batches of adenoviral vectors.


Classical and emergent cardiovascular disease risk factors in type 2 diabetics from the Vallecas area (DICARIVA study).
Type 2 diabetes mellitus (T2DM) is a major independent risk factor for cardiovascular disease (CVD) and a highly prevalent disease with a wide variety of associated metabolic disorders. To describe features and prevalence of altered CVD risk factors in a T2DM population: DIabetes CArdiovascular RIsk of VAllecas (DICARIVA) study. 735 adult Spanish patients of the Vallecas area with T2DM from the Infanta Leonor Hospital (Madrid, Spain) were included in the study. Age, disease time-evolution, anthropometric measurements, glycemia, glucated haemoglobin A1c (HbA1c), lipid/lipoprotein profile, total cholesterol/high density lipoprotein cholesterol (HDL-cholesterol), low density lipoprotein cholesterol (LDL-cholesterol)/HDL-cholesterol and triglycerides/HDL-cholesterol ratios, triglycerides-glucose index (TyG), fibrinogen, high sensitivity-c reactive protein (hs-CRP) and microalbuminuria were assessed. Mean, standard deviations, and percentile distributions were obtained in males, females and the whole T2DM population for classical and emergent CVD risk markers. Obesity was found in 45% of patients, while 60% had high cardiovascular risk according to waist circumference and conicity index. Total and LDL-cholesterol were at desirable and optimum levels, respectively, in 60% of patients. One third showed the conjoint presence of low HDL-cholesterol, high triglycerides and small and dense LDL. Increased levels of hs-CRP, hyperfibrinogenia and microalbuminuria were detected in 40%, 50% and 30% of patients, respectively. Age, body mass index, total cholesterol, hs-CRP and fibrinogen were higher while weight, conicity index, total cholesterol/HDL-cholesterol, LDL-cholesterol/HDL-cholesterol and triglycerides/HDL-cholesterol ratios, and microalbuminuria lower in women. According to TyG values 62% of patients suffered metabolic syndrome. Altered anthropometric and metabolic CVD risk factors were highly prevalent in the DICARIVA study. The CVD marker cut-off points obtained in some emergent markers seems relevant and would be employed for future early T2DM diagnoses strategy in order to reduce its high morbidity and mortality impact.


Characterization of human mesothelin transcripts in ovarian and pancreatic cancer.
Mesothelin is an attractive target for cancer immunotherapy due to its restricted expression in normal tissues and high level expression in several tumor types including ovarian and pancreatic adenocarcinomas. Three mesothelin transcript variants have been reported, but their relative expression in normal tissues and tumors has been poorly characterized. The goal of the present study was to clarify which mesothelin transcript variants are commonly expressed in human tumors. Human genomic and EST nucleotide sequences in the public databases were used to evaluate sequences reported for the three mesothelin transcript variants in silico. Subsequently, RNA samples from normal ovary, ovarian and pancreatic carcinoma cell lines, and primary ovarian tumors were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and nucleotide sequencing to directly identify expressed transcripts. In silico comparisons of genomic DNA sequences with available EST sequences supported expression of mesothelin transcript variants 1 and 3, but there were no sequence matches for transcript variant 2. Newly-derived nucleotide sequences of RT-PCR products from tissues and cell lines corresponded to mesothelin transcript variant 1. Mesothelin transcript variant 2 was not detected. Transcript variant 3 was observed as a small percentage of total mesothelin amplification products from all studied cell lines and tissues. Fractionation of nuclear and cytoplasmic RNA indicated that variant 3 was present primarily in the nuclear fraction. Thus, mesothelin transcript variant 3 may represent incompletely processed hnRNA. Mesothelin transcript variant 1 represents the predominant mature mRNA species expressed by both normal and tumor cells. This conclusion should be important for future development of cancer immunotherapies, diagnostic tests, and gene microarray studies targeting mesothelin.


IRF5 Is a Key Regulator of Macrophage Response to Lipopolysaccharide in Newborns.
Infections are a leading cause of mortality and morbidity in newborns. The high susceptibility of newborns to infection has been associated with a limited capacity to mount protective immune responses. Monocytes and macrophages are involved in the initiation, amplification, and termination of immune responses. Depending on cues received from their environment, monocytes differentiate into M1 or M2 macrophages with proinflammatory or anti-inflammatory and tissue repair properties, respectively. The purpose of this study was to characterize differences in monocyte to macrophage differentiation and polarization between newborns and adults. Monocytes from umbilical cord blood of healthy term newborns and from peripheral blood of adult healthy subjects were exposed to GM-CSF or M-CSF to induce M1 or M2 macrophages. Newborn monocytes differentiated into M1 and M2 macrophages with similar morphology and expression of differentiation/polarization markers as adult monocytes, with the exception of CD163 that was expressed at sevenfold higher levels in newborn compared to adult M1 macrophages. Upon TLR4 stimulation, newborn M1 macrophages produced threefold to sixfold lower levels of TNF than adult macrophages, while production of IL-1-β, IL-6, IL-8, IL-10, and IL-23 was at similar levels as in adults. Nuclear levels of IRF5, a transcription factor involved in M1 polarization, were markedly reduced in newborns, whereas the NF-κB and MAP kinase pathways were not altered. In line with a functional role for IRF5, adenoviral-mediated IRF5 overexpression in newborn M1 macrophages restored lipopolysaccharide-induced TNF production. Altogether, these data highlight a distinct immune response of newborn macrophages and identify IRF5 as a key regulator of macrophage TNF response in newborns.


Normal CA1 Place Fields but Discoordinated Network Discharge in a Fmr1-Null Mouse Model of Fragile X Syndrome.
Silence of FMR1 causes loss of fragile X mental retardation protein (FMRP) and dysregulated translation at synapses, resulting in the intellectual disability and autistic symptoms of fragile X syndrome (FXS). Synaptic dysfunction hypotheses for how intellectual disabilities like cognitive inflexibility arise in FXS predict impaired neural coding in the absence of FMRP. We tested the prediction by comparing hippocampus place cells in wild-type and FXS-model mice. Experience-driven CA1 synaptic function and synaptic plasticity changes are excessive in Fmr1-null mice, but CA1 place fields are normal. However, Fmr1-null discharge relationships to local field potential oscillations are abnormally weak, stereotyped, and homogeneous; also, discharge coordination within Fmr1-null place cell networks is weaker and less reliable than wild-type. Rather than disruption of single-cell neural codes, these findings point to invariant tuning of single-cell responses and inadequate discharge coordination within neural ensembles as a pathophysiological basis of cognitive inflexibility in FXS. VIDEO ABSTRACT.


Cytosolic and nuclear estrogen receptors in the isolated perfused rat liver.
The isolated perfused rat liver was investigated as a potential model to analyze binding of 17 beta-[3H]estradiol (E2) to cytosolic and nuclear estrogen receptors. Viability of the isolated perfused liver was monitored by measuring leakage of cytosolic enzymes into the perfusate. These studies indicated that the liver remained viable for at least a 90-min perfusion period although significant decreases in cytosolic estrogen receptor concentrations occurred during this perfusion period. Estrogen receptor loss was minimized by supplementing the red blood cell-free perfusion medium with 5 micrograms of insulin per ml. Uptake of [3H]E2 by hepatic cytosolic estrogen receptors of the isolated perfused liver was rapid as measured by partial purification of radiolabeled ligand receptor complexes after varying times of perfusion. Peak liver concentrations of receptor-bound E2 were achieved 15 min after the onset of perfusion when using livers from either male or female rats. After 15 min, radiolabeled cytosolic ligand receptor complexes decreased rapidly, reaching lowest concentrations at 60 min. Radiolabeled salt-extractable nuclear-binding sites increased up to 30 min and then decreased slightly between 30 and 90 min. Both 4S and 5S forms of nuclear binding sites were detected in the isolated perfused livers as evaluated by sedimentation analysis on 5 to 20% sucrose density gradients. Concentrations of radiolabeled cytosolic and nuclear receptors were greater in females than males at all perfusion periods examined when the initial concentration of [3H]E2 was 4 nm. Sex differences in receptor uptake were not as great when higher concentrations of [3H]E2 were added to the perfusion medium. These studies suggest that the isolated perfused liver is a suitable model to investigate short-term uptake of estrogens by cytosolic and nuclear receptors.


Induced pluripotent stem cell models of the genomic imprinting disorders Angelman and Prader-Willi syndromes.
Angelman syndrome (AS) and Prader-Willi syndrome (PWS) are neurodevelopmental disorders of genomic imprinting. AS results from loss of function of the ubiquitin protein ligase E3A (UBE3A) gene, whereas the genetic defect in PWS is unknown. Although induced pluripotent stem cells (iPSCs) provide invaluable models of human disease, nuclear reprogramming could limit the usefulness of iPSCs from patients who have AS and PWS should the genomic imprint marks be disturbed by the epigenetic reprogramming process. Our iPSCs derived from patients with AS and PWS show no evidence of DNA methylation imprint erasure at the cis-acting PSW imprinting center. Importantly, we find that, as in normal brain, imprinting of UBE3A is established during neuronal differentiation of AS iPSCs, with the paternal UBE3A allele repressed concomitant with up-regulation of the UBE3A antisense transcript. These iPSC models of genomic imprinting disorders will facilitate investigation of the AS and PWS disease processes and allow study of the developmental timing and mechanism of UBE3A repression in human neurons.


Risk factors for deterioration of erectile function: the Krimpen study.
This report from the Krimpen study explored the relationship between the determinants for worsening of erectile function in the open population. In Krimpen aan den IJssel (a municipality near Rotterdam), all men aged 50-75 years, without cancer of the prostate or the bladder and without a history of radical prostatectomy or neurogenic bladder disease, were invited to participate in June 1995. The response rate was 50%. The follow-up was until June 2004. At baseline a visit to a health centre for the measurement of urinalysis, height, weight and blood pressure was part of the ongoing study. During baseline and at the first follow-up, second follow-up and third follow-up, a self-administered booklet consisting of a compilation of validated questionnaires including the International Continence Society male sex questionnaire was completed. At the urology outpatient clinic, a urological workup was measured. All participants were asked to keep a frequency-volume chart for 3 days. A multivariate Cox-proportional hazard model was constructed to find the determinants of worsening of erectile function, correcting for age. Total follow-up time was 4948 person years consisting of 975 men. During follow-up, 441 events of worsening of erectile function occurred. Multivariate Cox-proportional hazard ratio analyses showed that body mass index (BMI), irritative lower urinary tract symptoms, diabetes mellitus, chronic obstructive pulmonary disease (COPD) and sexual inactivity were determinants with significant hazard ratios. In addition to age, determinants for a deterioration of erectile function based on multivariate longitudinal analyses are BMI, diabetes mellitus, COPD, sexual inactivity and irritative IPSS. The mechanism of various determinants is discussed.


Occurrence and distribution of Giardia species in wild rodents in Germany.
Giardiasis is an important gastrointestinal parasitic disease in humans and other mammals caused by the protozoan Giardia duodenalis. This species complex is represented by genetically distinct groups (assemblages A-H) with varying zoonotic potential and host preferences. Wild rodents can harbor potentially zoonotic assemblages A and B, and the rodent-specific assemblage G. Other Giardia spp. found in these animals are Giardia muris and Giardia microti. For the latter, only limited information on genetic typing is available. It has been speculated that wild rodents might represent an important reservoir for parasites causing human giardiasis. The aim of this study was to investigate the occurrence and distribution of Giardia spp. and assemblage types in wild rodents from different study sites in Germany. Screening of 577 wild rodents of the genera Apodemus, Microtus and Myodes, sampled at eleven study sites in Germany, revealed a high overall Giardia prevalence. Giardia species determination at the SSU rDNA gene locus revealed that Apodemus mice, depending on species, were predominantly infected with one of two distinct G. muris sequence types. Giardia microti was the predominant parasite species found in voles of the genera Microtus and Myodes. Only a few animals were positive for potentially zoonotic G. duodenalis. Subtyping at the beta-giardin (bg) and glutamine dehydrogenase (gdh) genes strongly supported the existence of different phylogenetic subgroups of G. microti that are preferentially harbored by distinct host species. The present study highlights the preference of G. muris for Apodemus, and G. microti for Microtus and Myodes hosts and argues for a very low prevalence of zoonotic G. duodenalis assemblages in wild rodents in Germany. It also provides evidence that G. muris and G. microti subdivide into several phylogenetically distinguishable subgroups, each of which appears to be preferentially harbored by species of a particular rodent host genus. Finally, the study expands the database of sequences relevant for sequence typing of G. muris and G. microti isolates which will greatly help future analyses of these parasites' population structure.


Identification of a polyI:C-inducible membrane protein that participates in dendritic cell-mediated natural killer cell activation.
In myeloid dendritic cells (mDCs), TLR3 is expressed in the endosomal membrane and interacts with the adaptor toll/interleukin 1 receptor homology domain-containing adaptor molecule 1 (TICAM-1; TRIF). TICAM-1 signals culminate in interferon (IFN) regulatory factor (IRF) 3 activation. Co-culture of mDC pretreated with the TLR3 ligand polyI:C and natural killer (NK) cells resulted in NK cell activation. This activation was triggered by cell-to-cell contact but not cytokines. Using expression profiling and gain/loss-of-function analyses of mDC genes, we tried to identify a TICAM-1-inducing membrane protein that participates in mDC-mediated NK activation. Of the nine candidates screened, one contained a tetraspanin-like sequence and satisfied the screening criteria. The protein, referred to as IRF-3-dependent NK-activating molecule (INAM), functioned in both the mDC and NK cell to facilitate NK activation. In the mDC, TICAM-1, IFN promoter stimulator 1, and IRF-3, but not IRF-7, were required for mDC-mediated NK activation. INAM was minimally expressed on NK cells, was up-regulated in response to polyI:C, and contributed to mDC-NK reciprocal activation via its cytoplasmic tail, which was crucial for the activation signal in NK cells. Adoptive transfer of INAM-expressing mDCs into mice implanted with NK-sensitive tumors caused NK-mediated tumor regression. We identify a new pathway for mDC-NK contact-mediated NK activation that is governed by a TLR signal-derived membrane molecule.


A picture tells a thousand words: A content analysis of concussion-related images online.
Recently image-sharing social media platforms have become a popular medium for sharing health-related images and associated information. However within the field of sports medicine, and more specifically sports related concussion, the content of images and meta-data shared through these popular platforms have not been investigated. The aim of this study was to analyse the content of concussion-related images and its accompanying meta-data on image-sharing social media platforms. We retrieved 300 images from Pinterest, Instagram and Flickr by using a standardised search strategy. All images were screened and duplicate images were removed. We excluded images if they were: non-static images; illustrations; animations; or screenshots. The content and characteristics of each image was evaluated using a customised coding scheme to determine major content themes, and images were referenced to the current international concussion management guidelines. From 300 potentially relevant images, 176 images were included for analysis; 70 from Pinterest, 63 from Flickr, and 43 from Instagram. Most images were of another person or a scene (64%), with the primary content depicting injured individuals (39%). The primary purposes of the images were to share a concussion-related incident (33%) and to dispense education (19%). For those images where it could be evaluated, the majority (91%) were found to reflect the Sports Concussion Assessment Tool 3 (SCAT3) guidelines. The ability to rapidly disseminate rich information though photos, images, and infographics to a wide-reaching audience suggests that image-sharing social media platforms could be used as an effective communication tool for sports concussion. Public health strategies could direct educative content to targeted populations via the use of image-sharing platforms. Further research is required to understand how image-sharing platforms can be used to effectively relay evidence-based information to patients and sports medicine clinicians.


Secretion of type III effectors into host cells in real time.
Type III secretion (T3S) systems are key features of many gram-negative bacteria that translocate T3S effector proteins directly into eukaryotic cells. There, T3S effectors exert many effects, such as cellular invasion or modulation of host immune responses. Studying spatiotemporal orchestrated secretion of various effectors has been difficult without disrupting their functions. Here we developed a new approach using Shigella flexneri T3S as a model to investigate bacterial translocation of individual effectors via multidimensional time-lapse microscopy. We demonstrate that direct fluorescent labeling of tetracysteine motif-tagged effectors IpaB and IpaC is possible in situ without loss of function. Studying the T3S kinetics of IpaB and IpaC ejection from individual bacteria, we found that the entire pools of IpaB and IpaC were released concurrently upon host cell contact, and that 50% of each effector was secreted in 240 s. This method allows an unprecedented analysis of the spatiotemporal events during T3S.


Pathogenesis of dermonecrosis induced by venom of the spitting cobra, Naja nigricollis: An experimental study in mice.
The pathogenesis of dermonecrosis induced by the venom of the African spitting cobra Naja nigricollis was investigated in a mouse model. Intradermal injection of venom induced a macroscopic necrotic lesion. Histological examination revealed early edema of the dermis, followed by blistering, loss of skin appendages and reduction in cellularity. By 24 h, necrosis of the dermis was evident, sections of epidermis were lost, and a fibrinoid hyaline material filled the damaged areas. Abundant inflammatory infiltrate was present in the hypodermis and basal dermis, and there was an increment in the expression of matrix metalloproteinases (MMPs). Thrombi were observed in blood vessels. Abundant cells were present in the dermis by 7 days. By 14 and 28 days, re-epithelization had occurred, collagen was widespread in the dermis, and few skin appendages were present. The RP-HPLC fractions that reproduced the necrotic activity were composed of low molecular mass cytotoxins of the three-finger toxin family and, to a lesser extent, of phospholipases A2 (PLA2). Inhibition of PLA2 of venom by p-bromophenacyl bromide did not reduce the area of necrosis, but modified the appearance of necrotic regions. Depletion of neutrophils and inhibition of venom metalloproteinases and tissue MMPs did not affect dermonecrosis. IgG and F(ab')2 antivenoms were effective in the neutralization of dermonecrosis when incubated with venom prior to injection. However, when antivenoms were administered immediately after venom injection, dermonecrosis was reduced only to a partial extent, underscoring the difficulties in neutralizing this effect with antivenoms.


DNA polymerases beta and lambda mediate overlapping and independent roles in base excision repair in mouse embryonic fibroblasts.
Base excision repair (BER) is a DNA repair pathway designed to correct small base lesions in genomic DNA. While DNA polymerase beta (pol beta) is known to be the main polymerase in the BER pathway, various studies have implicated other DNA polymerases in back-up roles. One such polymerase, DNA polymerase lambda (pol lambda), was shown to be important in BER of oxidative DNA damage. To further explore roles of the X-family DNA polymerases lambda and beta in BER, we prepared a mouse embryonic fibroblast cell line with deletions in the genes for both pol beta and pol lambda. Neutral red viability assays demonstrated that pol lambda and pol beta double null cells were hypersensitive to alkylating and oxidizing DNA damaging agents. In vitro BER assays revealed a modest contribution of pol lambda to single-nucleotide BER of base lesions. Additionally, using co-immunoprecipitation experiments with purified enzymes and whole cell extracts, we found that both pol lambda and pol beta interact with the upstream DNA glycosylases for repair of alkylated and oxidized DNA bases. Such interactions could be important in coordinating roles of these polymerases during BER.


Cell viability effects of triamcinolone acetonide and preservative vehicle formulations.
To assess the effect of triamcinolone acetonide and preservative vehicle formulations on human retinal pigment epithelium (ARPE19) cells over a range of concentrations. Triamcinolone acetonide, in its trade and preservative free formulations, along with the preservative vehicle were added to ARPE19 cell cultures in various concentrations (0.01-1.0 mg/ml). Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at day 5 after exposure. Functionality of the cultured ARPE19 cell line was confirmed by exposure to a previously characterised toxic agent, tamoxifen. The ARPE19 cell line behaved as predicted with exposure to tamoxifen. All formulations caused significant reductions in ARPE19 cell viability at the highest concentrations (1.0 mg/ml for triamcinolone preparations and undiluted vehicle). Cell viability was reduced to the greatest degree in trade formulation triamcinolone, less so by the vehicle, and least by preservative free triamcinolone. At lower concentrations no significant effect on cell viability was observed, although cell viability was found to be inversely proportional to increasing concentration of all tested reagents Both the trade and preservative free formulations of triamcinolone acetonide as well as the vehicle result in cell loss at in vitro concentrations of 1 mg/ml. Although this represents theoretical vitreous concentrations achieved with current widespread therapeutic use it probably does not indicate the actual exposure of cells in their biological milieu. That cell viability was reduced most in the trade formulation suggests a possible potentiated inhibitory toxic effect of triamcinolone acetonide and vehicle at higher concentrations.


Genetic signatures of exceptional longevity in humans.
Healthy aging is thought to reflect the combined influence of environmental factors (lifestyle choices) and genetic factors. To explore the genetic contribution, we undertook a genome-wide association study of exceptional longevity (EL) in 1055 centenarians and 1267 controls. Using these data, we built a genetic model that includes 150 single-nucleotide polymorphisms (SNPs) and found that it could predict EL with 77% accuracy in an independent set of centenarians and controls. Further in silico analysis revealed that 90% of centenarians can be grouped into 19 clusters characterized by different combinations of SNP genotypes-or genetic signatures-of varying predictive value. The different signatures, which attest to the genetic complexity of EL, correlated with differences in the prevalence and age of onset of age-associated diseases (e.g., dementia, hypertension, and cardiovascular disease) and may help dissect this complex phenotype into subphenotypes of healthy aging.


Modern optics in exceptionally preserved eyes of Early Cambrian arthropods from Australia.
Despite the status of the eye as an "organ of extreme perfection", theory suggests that complex eyes can evolve very rapidly. The fossil record has, until now, been inadequate in providing insight into the early evolution of eyes during the initial radiation of many animal groups known as the Cambrian explosion. This is surprising because Cambrian Burgess-Shale-type deposits are replete with exquisitely preserved animals, especially arthropods, that possess eyes. However, with the exception of biomineralized trilobite eyes, virtually nothing is known about the details of their optical design. Here we report exceptionally preserved fossil eyes from the Early Cambrian (∼ 515 million years ago) Emu Bay Shale of South Australia, revealing that some of the earliest arthropods possessed highly advanced compound eyes, each with over 3,000 large ommatidial lenses and a specialized 'bright zone'. These are the oldest non-biomineralized eyes known in such detail, with preservation quality exceeding that found in the Burgess Shale and Chengjiang deposits. Non-biomineralized eyes of similar complexity are otherwise unknown until about 85 million years later. The arrangement and size of the lenses indicate that these eyes belonged to an active predator that was capable of seeing in low light. The eyes are more complex than those known from contemporaneous trilobites and are as advanced as those of many living forms. They provide further evidence that the Cambrian explosion involved rapid innovation in fine-scale anatomy as well as gross morphology, and are consistent with the concept that the development of advanced vision helped to drive this great evolutionary event.


A canine model of femoral head osteonecrosis induced by an ethanol injection navigated by a novel template.
There is no consensus on how to establish models of osteonecrosis of the femoral head (ONFH) in large mammals. The aim of this study was to investigate the effectiveness of a novel canine model of ONFH, induced by a navigated injection of absolute ethanol. Using three-dimensional reconstruction and rapid prototyping manufacturing techniques, a new template was designed and processed to navigate the ethanol injection. The femoral heads of 18 adult dogs were injected with ethanol. Macroscopic, X-ray and histological examinations were performed at 3, 6, and 9 weeks after the operation. Further, computed tomography (CT), magnetic resonance imaging (MRI), and radionuclide scans were performed 6 weeks postoperatively. Three weeks after the operation, the femoral heads showed evidence of osteonecrosis including increasing numbers of empty lacunae, decreased hematopoietic cells, and destroyed adipose tissue in the medullary cavity, which increased in severity at the subsequent follow-up evaluations at 6 and 9 weeks. Fractured trabeculae and fibrous tissue were noted 9 weeks postoperatively. Image analysis also revealed evidence of osteonecrosis, such as several osteopenic areas with sclerotic rims on the X-ray, several areas of low bone mineral density with sclerosis on the CT scan, increased uptake of the nuclide species in MRI, and an inhomogeneous long T2 signal on the radioisotopic images. Ethanol injection navigated by our novel template was successful in establishing a canine model of ONFH. This model can be used to test new treatment modalities for human ONFH.


Forensic facial comparison: issues of admissibility in the development of novel analytical technique.
Much contemporary debate in forensic science concerns validity and admissibility of scientific evidence in court. In this paper, three current approaches to facial identification--image superimposition, photogrammetry, and morphological analysis--are considered with regard to criteria for scientific evidence in the United States, and England, and Wales. The aim of the paper is to assess the extent to which facial image comparison meets criteria of admissibility in these jurisdictions. The method used is a comparative evaluation of the methods of facial image comparison and their underlying premises against the range of admissibility criteria reported in court rulings and relevant judicial and scientific inquiries in the United States and the United Kingdom. While the techniques of facial image comparison are generally accepted within their practitioner communities, they are not tested, and their error rates are unknown. On that basis, the methods of facial image comparison would appear not to meet the anticipated standards. They are, nevertheless, admitted in court in the United States, and England, and Wales. This paper concludes that further research in science and law will be necessary to more definitively establish admissibility of facial image comparison evidence, as it will for other nascent and novel methods that are potentially influential in court proceedings.


Blood vascular organization of the rat carotid body: a scanning electron microscopic study of corrosion casts.
The blood vascular bed of the rat carotid body was reproduced with methacrylate and observed under the scanning electron microscope. The carotid body received the proper carotid body artery from the common carotid body artery, which arose from the external carotid or occipital artery and gave off subsidiary branches to the tissues near the carotid body. The proper carotid body artery divided in the carotid body, ultimately breaking up into thick (main) or thin (subsidiary) arterial terminals to form the vascular plexus of the carotid body. This plexus contained both thick and thin capillaries. The thick capillaries arose from the thick and thin arterial terminals and formed the basic capillary network of the carotid body. The thin capillaries were only subsidiary, intercalated among the thick capillaries. A few accessory twigs of the proper carotid body artery passed through the carotid body and supplied the adipose and other tissues around the carotid body. Many venules arose from the thick capillaries of the carotid body and were collected into rostral and caudal efferent veins. These efferent veins received the veins from the tissues adjacent to the carotid body, and drained into the internal jugular vein. No arterio-venous anastomosis was found in, on or around the carotid body. The common carotid body artery and its subsidiary branches showed, at their origins, marked constrictions indicative of the arterial cushions, though the proper carotid body artery and its accessory twigs were not provided with such clear constrictions. These findings suggest that the inflow of blood into the common carotid body artery may be regulated by its constriction, especially of its arterial cushion, and that the subsidiary branches of the common carotid body artery and the accessory twigs of the proper carotid body artery may act as bypass-routes to eliminate the excessive inflow of blood into the carotid body. It is considered that the thin arterial terminals and thin capillaries may act as buffer channels to homogenize the blood flow within the carotid body.


Physiological analysis of the stringent response elicited in an extreme thermophilic bacterium, Thermus thermophilus.
Guanosine tetraphosphate (ppGpp) is a key mediator of stringent control, an adaptive response of bacteria to amino acid starvation, and has thus been termed a bacterial alarmone. Previous X-ray crystallographic analysis has provided a structural basis for the transcriptional regulation of RNA polymerase activity by ppGpp in the thermophilic bacterium Thermus thermophilus. Here we investigated the physiological basis of the stringent response by comparing the changes in intracellular ppGpp levels and the rate of RNA synthesis in stringent (rel(+); wild type) and relaxed (relA and relC; mutant) strains of T. thermophilus. We found that in wild-type T. thermophilus, as in other bacteria, serine hydroxamate, an amino acid analogue that inhibits tRNA(Ser) aminoacylation, elicited a stringent response characterized in part by intracellular accumulation of ppGpp and that this response was completely blocked in a relA-null mutant and partially blocked in a relC mutant harboring a mutation in the ribosomal protein L11. Subsequent in vitro assays using ribosomes isolated from wild-type and relA and relC mutant strains confirmed that (p)ppGpp is synthesized by ribosomes and that mutation of RelA or L11 blocks that activity. This conclusion was further confirmed in vitro by demonstrating that thiostrepton or tetracycline inhibits (p)ppGpp synthesis. In an in vitro system, (p)ppGpp acted by inhibiting RNA polymerase-catalyzed 23S/5S rRNA gene transcription but at a concentration much higher than that of the observed intracellular ppGpp pool size. On the other hand, changes in the rRNA gene promoter activity tightly correlated with changes in the GTP but not ATP concentration. Also, (p)ppGpp exerted a potent inhibitory effect on IMP dehydrogenase activity. The present data thus complement the earlier structural analysis by providing physiological evidence that T. thermophilus does produce ppGpp in response to amino acid starvation in a ribosome-dependent (i.e., RelA-dependent) manner. However, it appears that in T. thermophilus, rRNA promoter activity is controlled directly by the GTP pool size, which is modulated by ppGpp via inhibition of IMP dehydrogenase activity. Thus, unlike the case of Escherichia coli, ppGpp may not inhibit T. thermophilus RNA polymerase activity directly in vivo, as recently proposed for Bacillus subtilis rRNA transcription (L. Krasny and R. L. Gourse, EMBO J. 23:4473-4483, 2004).


Sterol intermediates from cholesterol biosynthetic pathway as liver X receptor ligands.
The liver X receptors (LXRs) are ligand-activated transcription factors that regulate the expression of genes controlling lipid metabolism. Oxysterols bind LXRs with high affinity in vitro and are implicated as ligands for the receptor. We showed previously that accumulation of selected dietary sterols, in particular stigmasterol, is associated with activation of LXR in vivo. In the course of the defining of structural features of stigmasterol that confer LXR agonist activity, we determined that the presence of an unsaturated bond in the side chain of the sterol was necessary and sufficient for activity, with the C-24 unsaturated cholesterol precursor sterols desmosterol and zymosterol exerting the largest effects. Desmosterol failed to increase expression of the LXR target gene, ABCA1, in LXRalpha/beta-deficient mouse fibroblasts, but was fully active in cells lacking cholesterol 24-, 25-, and 27-hydroxylase; thus, the effect of desmosterol was LXR-dependent and did not require conversion to a side chain oxysterol. Desmosterol bound to purified LXRalpha and LXRbeta in vitro and supported the recruitment of steroid receptor coactivator 1. Desmosterol also inhibited processing of the sterol response element-binding protein-2 and reduced expression of hydroxymethylglutaryl-CoA reductase. These observations are consistent with specific intermediates in the cholesterol biosynthetic pathway regulating lipid homeostasis through both the LXR and sterol response element-binding protein pathways.


On the origin of the driving force in the Marangoni propelled gas bubble trapping mechanism.
Gas bubbles can be trapped and then manipulated with laser light. In this report, we propose the detailed optical trapping mechanism of gas bubbles confined inside a thin light-absorbing liquid layer between two glass plates. The necessary condition of bubble trapping in this case is the direct absorption of light by the solution containing a dye. Due to heat release, fluid whirls propelled by the surface Marangoni effect at the liquid/gas interface emerge and extend to large distances. We report the experimental microscopic observation of the origin of whirls at an initially flat liquid/air interface as well as at the curved interface of a liquid/gas bubble and support this finding with advanced numerical simulations using the finite element method within the COMSOL Multiphysics platform. The simulation results were in good agreement with the observations, which allowed us to propose a simple physical model for this particular trapping mechanism, to establish the origin of forces attracting bubbles toward a laser beam and to predict other phenomena related to this effect.


Ninety-six hours ordeal of priapism induced by paroxetine--A case report and literature review.
Priapism is a rare but severe medical condition of sustained and painful erection of penis in the absence of any sexual stimulation, in which the penis does not return to a flaccid state. It is considered to be a medical emergency because without treatment it can lead to permanent damage and fibrosis of penis and in the long run to impotency. Albeit that there is no uniform consensus regarding the duration of sustained erection, an erection lasting more than 4 h is generally considered as an emergency which needs immediate medical attention and care. Priapism is commonly associated with iatrogenic, pharmacologic, underlying medical, or traumatic causes. In this report, we present the case of a 42-year-old African American man who developed priapism after three weeks of therapy with paroxetine which lasted for more than 96 h before coming to the attention of his health-care providers. This case is unique in that there are no reports in literature of an erection lasting for such a long duration following therapy with paroxetine. The objective of this report is to highlight the importance of recognizing the possibility of priapism with selective serotonin reuptake inhibitors in general and paroxetine in particular since this condition is not commonly seen in clinical practice to be associated with selective serotonin reuptake inhibitors and may go unrecognized. Also, potential biological mechanisms involved in the development of paroxetine-induced priapism are presented.


The dimensional characteristics of smooth muscle in rat blood vessels. A computer-assisted analysis.
This investigation was undertaken to provide precise information about the dimensional characteristics of vascular smooth muscle cells as related to their paracellular matrix. The representative types of vessels were fixed at the mean blood pressures of adult male Wistar rats. The shapes, positions of the nucleus, linear dimensions, volumes, and orientation within the vessel wall were determined by a computer-assisted reconstruction of the cells from serial sections. Wall-to-lumen and cellular-to-paracellular ratios also were assessed. The smooth muscle cells were elongate, but whereas some are spindle shaped, most are not, and may be shaped like flattened triangles, paddles, boomerangs, or hourglasses, and in addition, any one of these shapes may be forked. The nucleus tended to be in the largest part of the cell, wherever that region occurred. Thus, the majority of the nuclei (61%) were not centrally located, but overlapped the middle and end thirds of the elongate cells. Of the three arteries investigated, the muscular type tail artery had cells with volumes two to three times larger (P less than 0.01) than cells in a musculoelastic (femoral) or elastic (mesenteric) artery, and six times larger (P less than 0.01) than those of the portal vein. Therefore, the smooth muscle cells of the vein were significantly smaller than those in any artery (P less than 0.01). The smooth muscle cells were aligned at a steeper angle in the vessel wall (15 degrees +/- 2 degrees) of the muscular artery than in those with more elastic tissue (9 degrees +/- 2 degrees), with a higher percentage of circumferential cells in the latter. The wall-to-lumen ratios decreased as the relative amount of paracellular matrix, particularly elastic tissue, increased in the three arteries. Therefore, irregularly shaped cells, with the nucleus in the thickest region, and having characteristic cell volumes depending on the type of vessel, form the vascular smooth muscle tissue. These factors are relevant if stereology, or measuring from two dimensions, is used to estimate size characteristics in cardiovascular disease such as hypertension. In addition, the optimum angle at which vascular strips are cut would vary, for example, when used in testing pharmacological agents.


Antioxidant activities of curcumin and ascorbyl dipalmitate nanoparticles and their activities after incorporation into cellulose-based packaging films.
Curcumin (Ccm) and ascorbyl dipalmitate (ADP) nanoparticles (NPs) with average sizes of ∼50 and ∼80 nm, respectively, were successfully produced by rapid expansion of subcritical solutions into liquid solvents (RESOLV). Pluronic F127 was employed as a stabilizer for both Ccm- and ADP-NPs in an aqueous receiving solution. Antioxidant activities of the Ccm-NPs and ADP-NPs were subsequently investigated using four assays, including 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, ABTS radical cation decolorization, β-carotene bleaching, and ferric reducing antioxidant power. Ccm-NPs and ADP-NPs showed higher antioxidant activities than those of Ccm and ADP. Ccm-NPs yielded higher antioxidant activities than those of Ccm in ethanol and water (Ccm-EtOH and Ccm-H(2)O), respectively. ADP-NPs yielded lower antioxidant activities than that of ADP in ethanol (ADP-EtOH) but higher activities than that of ADP in water (ADP-H(2)O). Moreover, incorporation of Ccm-NPs and ADP-NPs into cellulose-based films indicated that Ccm-NPs and ADP-NPs significantly enhanced the antioxidant activities of Ccm and ADP (p < 0.05). Our results show that the environmentally benign supercritical CO(2) technique should be generally applicable to NP fabrication of other important bioactive ingredients, especially in liquid form. In addition, we suggest that Ccm-NPs and ADP-NPs can be used to reduce the dosage of Ccm and ADP and improve their bioavailability, and thus merit further investigation for antioxidant packaging film and coating applications.


Prospective study of matrix metalloproteinase-9 and risk of myocardial infarction and stroke in older men and women.
The endopeptidase matrix metalloproteinase-9 (MMP-9) is implicated in atherosclerotic plaque rupture. We investigate prospective associations between MMP-9 and MI or stroke in an older general population cohort, accounting for established and novel cardiovascular risk factors. Baseline serum MMP-9 was measured in incident MI (n=368) and stroke (n=299) cases and two controls per case, 'nested' in prospective studies of 4252 men and 4286 women aged 60-79 years, sampled from General Practices in Britain in 1998-2000, with 7-year follow-up for fatal and non-fatal MI and stroke. Geometric mean MMP-9 was 528 ng/mL (IQR 397, 743) in MI cases compared to 501 ng/mL (IQR 370, 743) in controls, p=0.10. Participants in the top compared to bottom third of MMP-9 levels had an age-adjusted odds ratio for MI of 1.53 (95% CI 1.09, 2.13), which attenuated to 1.18 (95% CI 0.81, 1.70) after adjustment for established and novel cardiovascular risk factors. There was weak evidence that OR differed according to pre-existing CVD; the OR for MI in 187 participants with pre-existing CVD was 2.20 (1.04, 4.64) and 1.24 (0.84, 1.82) in 715 participants without (LR test for interaction p=0.06). Geometric mean MMP-9 levels were higher in stroke cases than controls; 522ng/mL (IQR 363, 673) vs 487 (IQR 393, 704), p=0.045; however adjustments similarly attenuated the associations. While serum MMP-9 is univariately associated with risk of MI and stroke, it is not a strong independent risk marker for either.


Vascular responsiveness and cation exchange in insulin-dependent diabetes.
1. We measured forearm blood flow during brachial artery infusions of vasoconstrictors (angiotensin II and noradrenaline) and vasodilators (sodium nitroprusside and carbachol) in 16 healthy control subjects and in 18 patients with uncomplicated insulin-dependent diabetes mellitus. Erythrocyte Na+/Li+ countertransport and platelet Na+/H+ antiport activities were also measured. 2. The mean basal forearm vascular resistance was 22% lower in diabetic patients than in control subjects. The effects of each infusion on forearm vascular resistance were similar in diabetic patients and control subjects. 3. Erythrocyte Na+/Li+ countertransport activities were similar in diabetic patients and control subjects. Platelet Na+/H+ exchange (Vmax) was approximately 40% greater in diabetic patients than in control subjects, whereas the Km for Na+ was similar. 4. In diabetic patients, but not in control subjects, the responses to sodium nitroprusside and carbachol correlated inversely with Na+/Li+ countertransport in erythrocytes (rs = -0.76, P less than 0.001, and rs = -0.66, P less than 0.005, respectively), but not with Na+/H+ exchange in platelets.


The evolutionary basis for differences between the immune systems of man, mouse, pig and ruminants.
Studying the pathogenesis of an infectious disease like colibacillosis requires an understanding of the responses of target hosts to the organism both as a pathogen and as a commensal. The mucosal immune system constitutes the primary line of defence against luminal micro-organisms. The immunoglobulin-superfamily-based adaptive immune system evolved in the earliest jawed vertebrates, and the adaptive and innate immune system of humans, mice, pigs and ruminants co-evolved in common ancestors for approximately 300 million years. The divergence occurred only 100 mya and, as a consequence, most of the fundamental immunological mechanisms are very similar. However, since pressure on the immune system comes from rapidly evolving pathogens, immune systems must also evolve rapidly to maintain the ability of the host to survive and reproduce. As a consequence, there are a number of areas of detail where mammalian immune systems have diverged markedly from each other, such that results obtained in one species are not always immediately transferable to another. Thus, animal models of specific diseases need to be selected carefully, and the results interpreted with caution. Selection is made simpler where specific host species like cattle and pigs can be both target species and reservoirs for human disease, as in infections with Escherichia coli.


Activation of TRPV1 channels inhibits mechanosensitive Piezo channel activity by depleting membrane phosphoinositides.
Capsaicin is an activator of the heat-sensitive TRPV1 (transient receptor potential vanilloid 1) ion channels and has been used as a local analgesic. We found that activation of TRPV1 channels with capsaicin either in dorsal root ganglion neurons or in a heterologous expression system inhibited the mechanosensitive Piezo1 and Piezo2 channels by depleting phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and its precursor phosphatidylinositol 4-phosphate [PI(4)P] from the plasma membrane through Ca(2+)-induced phospholipase Cδ (PLCδ) activation. Experiments with chemically inducible phosphoinositide phosphatases and receptor-induced activation of PLCβ indicated that inhibition of Piezo channels required depletion of both PI(4)P and PI(4,5)P2. The mechanically activated current amplitudes decreased substantially in the excised inside-out configuration, where the membrane patch containing Piezo1 channels is removed from the cell. PI(4,5)P2 and PI(4)P applied to these excised patches inhibited this decrease. Thus, we concluded that Piezo channel activity requires the presence of phosphoinositides, and the combined depletion of PI(4,5)P2 and PI(4)P reduces channel activity. In addition to revealing a role for distinct membrane lipids in mechanosensitive ion channel regulation, these data suggest that inhibition of Piezo2 channels may contribute to the analgesic effect of capsaicin.


Is a specialist breathlessness service more effective and cost-effective for patients with advanced cancer and their carers than standard care? Findings of a mixed-method randomised controlled trial.
Breathlessness is common in advanced cancer. The Breathlessness Intervention Service (BIS) is a multi-disciplinary complex intervention theoretically underpinned by a palliative care approach, utilising evidence-based non-pharmacological and pharmacological interventions to support patients with advanced disease. We sought to establish whether BIS was more effective, and cost-effective, for patients with advanced cancer and their carers than standard care. A single-centre Phase III fast-track single-blind mixed-method randomised controlled trial (RCT) of BIS versus standard care was conducted. Participants were randomised to one of two groups (randomly permuted blocks). A total of 67 patients referred to BIS were randomised (intervention arm n = 35; control arm n = 32 received BIS after a two-week wait); 54 completed to the key outcome measurement. The primary outcome measure was a 0 to 10 numerical rating scale for patient distress due to breathlessness at two-weeks. Secondary outcomes were evaluated using the Chronic Respiratory Questionnaire, Hospital Anxiety and Depression Scale, Client Services Receipt Inventory, EQ-5D and topic-guided interviews. BIS reduced patient distress due to breathlessness (primary outcome: -1.29; 95% CI -2.57 to -0.005; P = 0.049) significantly more than the control group; 94% of respondents reported a positive impact (51/53). BIS reduced fear and worry, and increased confidence in managing breathlessness. Patients and carers consistently identified specific and repeatable aspects of the BIS model and interventions that helped. How interventions were delivered was important. BIS legitimised breathlessness and increased knowledge whilst making patients and carers feel 'not alone'. BIS had a 66% likelihood of better outcomes in terms of reduced distress due to breathlessness at lower health/social care costs than standard care (81% with informal care costs included). BIS appears to be more effective and cost-effective in advanced cancer than standard care. RCT registration at ClinicalTrials.gov NCT00678405 (May 2008) and Current Controlled Trials ISRCTN04119516 (December 2008).


Differential expression of novel metabolic and immunological biomarkers in oysters challenged with a virulent strain of OsHV-1.
Early lifestages of the Pacific oyster (Crassostrea gigas) are highly susceptible to infection by OsHV-1 μVar, but little information exists regarding metabolic or pathophysiological responses of larval hosts. Using a metabolomics approach, we identified a range of metabolic and immunological responses in oyster larvae exposed to OsHV-1 μVar; some of which have not previously been reported in molluscs. Multivariate analyses of entire metabolite profiles were able to separate infected from non-infected larvae. Correlation analysis revealed the presence of major perturbations in the underlying biochemical networks and secondary pathway analysis of functionally-related metabolites identified a number of prospective pathways differentially regulated in virus-exposed larvae. These results provide new insights into the pathogenic mechanisms of OsHV-1 infection in oyster larvae, which may be applied to develop disease mitigation strategies and/or as new phenotypic information for selective breeding programmes aiming to enhance viral resistance.


Eucaryotic genome evolution through the spontaneous duplication of large chromosomal segments.
There is growing evidence that duplications have played a major role in eucaryotic genome evolution. Sequencing data revealed the presence of large duplicated regions in the genomes of many eucaryotic organisms, and comparative studies have suggested that duplication of large DNA segments has been a continuing process during evolution. However, little experimental data have been produced regarding this issue. Using a gene dosage assay for growth recovery in Saccharomyces cerevisiae, we demonstrate that a majority of the revertant strains (58%) resulted from the spontaneous duplication of large DNA segments, either intra- or interchromosomally, ranging from 41 to 655 kb in size. These events result in the concomitant duplication of dozens of genes and in some cases in the formation of chimeric open reading frames at the junction of the duplicated blocks. The types of sequences at the breakpoints as well as their superposition with the replication map suggest that spontaneous large segmental duplications result from replication accidents. Aneuploidization events or suppressor mutations that do not involve large-scale rearrangements accounted for the rest of the reversion events (in 26 and 16% of the strains, respectively).


A variant ECE1 allele contributes to reduced pathogenicity of Candida albicans during vulvovaginal candidiasis.
Vulvovaginal candidiasis (VVC), caused primarily by the human fungal pathogen Candida albicans, results in significant quality-of-life issues for women worldwide. Candidalysin, a toxin derived from a polypeptide (Ece1p) encoded by the ECE1 gene, plays a crucial role in driving immunopathology at the vaginal mucosa. This study aimed to determine if expression and/or processing of Ece1p differs across C. albicans isolates and whether this partly underlies differential pathogenicity observed clinically. Using a targeted sequencing approach, we determined that isolate 529L harbors a similarly expressed, yet distinct Ece1p isoform variant that encodes for a predicted functional candidalysin; this isoform was conserved amongst a collection of clinical isolates. Expression of the ECE1 open reading frame (ORF) from 529L in an SC5314-derived ece1Δ/Δ strain resulted in significantly reduced vaginopathogenicity as compared to an isogenic control expressing a wild-type (WT) ECE1 allele. However, in vitro challenge of vaginal epithelial cells with synthetic candidalysin demonstrated similar toxigenic activity amongst SC5314 and 529L isoforms. Creation of an isogenic panel of chimeric strains harboring swapped Ece1p peptides or HiBiT tags revealed reduced secretion with the ORF from 529L that was associated with reduced virulence. A genetic survey of 78 clinical isolates demonstrated a conserved pattern between Ece1p P2 and P3 sequences, suggesting that substrate specificity around Kex2p-mediated KR cleavage sites involved in protein processing may contribute to differential pathogenicity amongst clinical isolates. Therefore, we present a new mechanism for attenuation of C. albicans virulence at the ECE1 locus.


Replication analysis for severe diabetic retinopathy.
The purpose of this study is to attempt to replicate the top single nucleotide polymorphism (SNP) associations from a previous genome-wide association study (GWAS) for the sight-threatening complications of diabetic retinopathy in an independent cohort of diabetic subjects from the Wisconsin Epidemiologic Study of Diabetic Retinopathy (WESDR). This study included 469 type 1 diabetic, Caucasian subjects from WESDR. Cases (n = 208) were defined by prior laser treatment for either proliferative diabetic retinopathy or diabetic macular edema. Controls (n = 261) were all other subjects in the cohort. Three hundred eighty-nine SNPs were tested for association using the Illumina GoldenGate custom array. A retinopathy-only subanalysis was conducted in 437 subjects by removing those with end-stage renal disease. Evaluation for association between cases and controls was conducted by using chi-square tests. A combined analysis incorporated the results from WESDR with the prior GWAS. No associations were significant at a genome-wide level. The analysis did identify SNPs that can be pursued in future replication studies. The top association was at rs4865047, an intronic SNP, in the gene CEP135 (P value 2.06 × 10(-5)). The top association from the subanalysis was at rs1902491 (P value 2.81 × 10(-5)), a SNP that sits upstream of the gene NPY2R. This study nominates several novel genetic loci that may be associated with severe diabetic retinopathy. In order to confirm these findings, replication and extension in additional cohorts will be necessary as susceptibility alleles for diabetic retinopathy appear to be of modest effect.


Integrated analysis of cytochrome P450 gene superfamily in the red flour beetle, Tribolium castaneum.
The functional and evolutionary diversification of insect cytochrome P450s (CYPs) shaped the success of insects. CYPs constitute one of the largest and oldest gene superfamilies that are found in virtually all aerobic organisms. Because of the availability of whole genome sequence and well functioning RNA interference (RNAi), the red flour beetle, Tribolium castaneum serves as an ideal insect model for conducting functional genomics studies. Although several T. castaneum CYPs had been functionally investigated in our previous studies, the roles of the majority of CYPs remain largely unknown. Here, we comprehensively analyzed the phylogenetic relationship of all T. castaneum CYPs with genes in other insect species, investigated the CYP6BQ gene cluster organization, function and evolution, as well as examined the mitochondrial CYPs gene expression patterns and intron-exon organization. A total 143 CYPs were identified and classified into 26 families and 59 subfamilies. The phylogenetic trees of CYPs among insects across taxa provided evolutionary insight for the genetic distance and function. The percentage of singleton (33.3%) in T. castaneum CYPs is much less than those in Drosophila melanogaster (52.5%) and Bombyx mori (51.2%). Most members in the largest CYP6BQ gene cluster may make contribution to deltamethrin resistance in QTC279 strain. T. castaneum genome encodes nine mitochondrial CYPs, among them CYP12H1 is only expressed in the final instar larval stage. The intron-exon organizations of these mitochondrial CYPs are highly diverse. Our studies provide a platform to understand the evolution and functions of T. castaneum CYP gene superfamily which will help reveal the strategies employed by insects to cope with their environment.


Intergenerational Transmission of DNA Methylation Signatures Associated with Early Life Stress.
Early life stress in humans (i.e. maltreatment, violence exposure, loss of a loved one) and in rodents (i.e. disrupted attachment or nesting, electric shock, restraint, predator odor) occurs during critical steps of neural circuit formation. ELS in humans is associated with increased risk for developmental psychopathology, including anxious and depressive phenotypes. The biological mechanisms underlying these potentially persistent maladaptive changes involve long-term epigenetic modifications, which have been suggested to be potentially transmissible to subsequent generations. DNA methylation is an epigenetic mechanism that modifies gene expression patterns in response to environmental challenges and influences mutation rates. It remains to be seen whether a functionally relevant fraction of DNA methylation marks can escape genome-wide erasures that occur in primordial germ cells and after fertilization within the zygote. Early life-stress-triggered changes in epigenetic mediated transmission of acquired behavioral traits among humans have been assessed mainly by targeting genes involved in the hypothalamic-pituitary-adrenal (HPA) axis, such as NR3C1 and FKBP5. Recently, researchers examining epigenetic transmission have begun to apply genome-wide approaches. In humans, reduced representation bisulfite sequencing (RRBS) was performed on peripheral samples that were obtained from individuals who were prenatally exposed to the "Dutch Hunger Winter", resulting in two Differentially Methylated Regions (DMRs) in INSR and CPTIA genes that were functionally, biologically and technically validated, and significantly associated with birth weights and LDL cholesterol levels in offspring. In rodents, non-genomic intergenerational transmission of anxiety which was associated with differentially methylated enhancers that were putatively involved in lipid signaling and synaptic/neurotransmission in hippocampal granule cells, was discovered also using RRBS. Finally, transgenerational transmission of altered behaviors was associated with sperm-derived microRNAs produced by ELS male mice. The field of epigenetic transmission is just beginning to enter the epigenomic era by using genome-wide analyses. Such approaches remain of strong interest to human studies, first in order to help to assess the relevance of the previous targeted studies, and second to discover new important epigenetic modifications of potential clinical importance. New discoveries may help to assess how transmittable the negative impact of stress may be to offspring. The latter may open doors for future treatments and resilience-promoting interventions, as well as new approaches to treat the effects of childhood trauma before the onset of psychiatric disorder.


Understanding volume-outcome relationships in nephrectomy and cystectomy for cancer: evidence from the UK Getting it Right First Time programme.
To investigate volume-outcome relationships in nephrectomy and cystectomy for cancer. Data were extracted from the UK Hospital Episodes Statistics database, which records data on all National Health Service (NHS) hospital admissions in the England. Data were included for a 5-year period (April 2013-March 2018 inclusive) and data on emergency and paediatric admissions were excluded. Data were extracted on the NHS trust and surgeon undertaking the procedure, the surgical technique used (open, laparoscopic or robot-assisted) and length of hospital stay during the procedure. This dataset was supplemented by data on mortality from the UK Office for National Statistics. A number of volume thresholds and volume measures were investigated. Multilevel modelling was used to adjust for hierarchy and confounding factors. Data were available for 18 107 nephrectomy and 6762 cystectomy procedures for cancer. There was little evidence of trust or surgeon volume influencing readmission rates or mortality. There was some evidence of shorter length of hospital stay for high-volume surgeons, although the volume measure and threshold used were important. We found little evidence that further centralization of nephrectomy or cystectomy for cancer surgery will improve the patient outcomes investigated. It may be that length of stay can be optimized though training and support for lower-volume centres, rather than further centralization.


Diagnostic role of neutrophil-lymphocyte ratio in oral cavity cancers.
In this study, we aimed to evaluate the diagnostic role of neutrophil-lymphocyte ratio (NLR) and other hemogram parameters in differentiating nonmalignant oral cavity lesions from oral cavity cancers. Ninety-five patients who were performed oral cavity biopsy between the years 2013 and 2015 were retrospectively reviewed. The control group comprised consecutive 70 patients who underwent septoplasty/septorhinoplasty procedures. Inclusion criteria were to be available with common blood count (CBC) just prior to procedure. The hemogram parameters including NLR were compared between the groups. Lymphocyte count and mean platelet volume (MPV) were found to be significantly decreased in oral cavity squamous cell carcinoma (SCC) and in other oral cavity cancers (OCCs) compared with benign oral cavity lesions and control group. In contrast, NLR revealed significantly higher in OCCs and in oral SCC compared with nonmalignant oral cavity lesions and control group. The receiver operating characteristics curve analysis suggested cutoff value of 2.88 for NLR in predicting malignancy [area under curve (AUC) 0.756, sensitivity 51%, specificity 88%]. NLR was first shown to be significantly elevated in oral cavity cancers and in oral cavity SCC in this study. In our opinion, NLR may be helpful in identifying the oral cavity lesions at high risk for harboring malignancy.


Esophageal, Gastric, and Duodenal Histologic Findings in Patients with Feeding Difficulties.
Currently, there are inconsistencies in the recommendations of when to obtain an esophagogastroduodenoscopy (EGD) in children with feeding difficulties. The aim of our study was to identify EGD findings in patients presenting to a large, outpatient feeding program. Additionally, we investigated the presence of any relationship between abnormal pathology seen on biopsies (inflammation) and symptoms of feeding intolerance such as vomiting, gagging, retching, or abdominal pain. Retrospective analysis of electronic medical records (EMRs) was conducted for all new patients aged 0-17 years presenting to the Multidisciplinary Feeding Clinic. Three hundred and thirty patients (50.2%) had an EGD with complete biopsies. Of these 330 patients, biopsies revealed esophagitis in 40%, gastritis in 33.6%, and duodenitis in 15.2%. Overall, 61.21% had an abnormal pathology in at least one site. We found that children with feeding disorders commonly have esophagitis, gastritis, and/or duodenitis and that symptoms are poor predictors of pathology. This study underscores the importance of gastrointestinal evaluation as part of a multidisciplinary evaluation in patients with feeding difficulties.


Particulate matter inhalation during hay storing activity induces systemic inflammation and platelet aggregation.
The aim of this study was to investigate possible pathomechanisms behind the cardiovascular morbidity caused by inhalation of particulate matter (PM(10)). For that purpose, healthy volunteers were exposed to high PM(10) concentrations during a 2 h hay storing activity. Blood was drawn in the evening before and after PM(10) exposure and in the morning and evening of the day after exposure. The leukocyte count increased after PM(10) exposure with an initial increase of segmented neutrophils followed by banded forms. C-reactive protein increased over time. Fibrinogen and plasma viscosity became increased in the evening of the day after PM(10) exposure. Platelet aggregation was increased in the evening after PM(10) exposure. At the same time von Willebrand factor and factor VIII were increased, reflecting endothelial activation. These results confirm that acute inhalative exposure to high PM(10) concentrations during hay storage activity leads to a systemic inflammatory reaction, endothelial activation, and platelet aggregation.


Effects of cardiorespiratory fitness on healthcare utilization.
Prospective study examining the relationship between cardiorespiratory fitness level and incidence of medical treatments during a 1-yr period before each of two examinations. A subset was also evaluated to assess whether improvement in fitness affected incidence of treatments. Part I: Six thousand six hundred seventy-nine healthy male subjects underwent medical examinations on two occasions, including a maximal exercise test. Division of subjects by fitness into quartiles (Q1 = low fitness through Q4 = high fitness) revealed an inverse relationship between fitness and outcome measures. Men in the low-fitness group had more office visits and overnight hospital stays than men in the high-fitness group (3.5% (Q1) vs 1.6% (Q4) men had 10+ office visits, and 10% (Q1) vs 5.0% (Q4) men had overnight hospital stays, P < 0.0001). These differences held after adjustment for potential confounding variables (age, follow-up yr, blood pressure, cholesterol, and smoking). Part II: Subjects in this subset (N = 2974) were evaluated to compare overnight hospital stays between low-fit men who remained low fit at the second examination, and low-fit men who became fit by the second examination. This cohort was divided into fitness tertiles (T1 = low fitness through T3 = high fitness). Those who improved their fitness by the time of the second examination had a decreased number of overnight hospital stays, compared with those who remained unfit at the time of the second examination (10.2% (T1 at second visit) vs 5.9% (T3 at second visit) had overnight hospital stays, P < 0.03). Men who maintain or become fit are less likely to have physician visits or overnight hospital stays, compared with men who are unfit.


A comparison of facial emotion processing in neurological and psychiatric conditions.
Patients suffering from various neurological and psychiatric disorders show different levels of facial emotion recognition (FER) impairment, sometimes from the early phases of the disease. Investigating the relative severity of deficits in FER across different clinical and high-risk populations has potential implications for the diagnosis and treatment of these diseases, and could also allow us to understand the neurobiological mechanisms of emotion perception itself. To investigate the role of the dopaminergic system and of the frontotemporal network in FER, we reanalyzed and compared data from four of our previous studies investigating FER performance in patients with frontotemporal dysfunctions and/or dopaminergic system abnormalities at different stages. The performance of patients was compared to the performance obtained by a specific group of matched healthy controls using Cohen's d effect size. We thus compared emotion and gender recognition in patients with frontotemporal dementia (FTD), amnestic mild cognitive impairment (aMCI), Alzheimer's disease (AD) at the mild dementia stage, major depressive disorder, Parkinson's disease treated by l-DOPA (PD-ON) or not (PD-OFF), remitted schizophrenia (SCZ-rem), first-episode schizophrenia treated by antipsychotic medication (SCZ-ON), and drug-naïve first-episode schizophrenia (SCZ-OFF), as well as in unaffected siblings of patients with schizophrenia (SIB). The analyses revealed a pattern of differential impairment of emotion (but not gender) recognition across pathological conditions. On the one hand, dopaminergic medication seems not to modify the moderate deficits observed in SCZ and PD groups (ON vs. OFF), suggesting that the deficit is independent from the dopaminergic system. On the other hand, the observed increase in effect size of the deficit among the aMCI, AD, and FTD groups (and also among the SIB and SCZ-rem groups) suggests that the deficit is dependent on neurodegeneration of the frontotemporal neural networks. Our transnosographic approach combining clinical and high-risk populations with the impact of medication provides new information on the trajectory of impaired emotion perception in neuropsychiatric conditions, and on the role of the dopaminergic system and the frontotemporal network in emotion perception.


Accounting for actions and omissions: a discourse analysis of student nurse accounts of responding to instances of poor care.
To explore how nursing students account for decisions to report or not report poor care witnessed on placement and to examine the implications of findings for educators. Concern has been raised about the extent to which cases of poor care go unreported. Failure to report cases may have serious consequences for patient safety. Semistructured interviews were conducted with 13 undergraduate students at a UK university during 2013. They were asked to consider their response to episodes of poor practice witnessed on placement. Data were transcribed verbatim and categorized according to whether or not students reported concerns. Cases were analysed in accordance with Potter and Wetherall's version of discourse analysis to identify the discursive strategies used to account for decisions to report or not report poor practice. Participants took care to present themselves in a positive light regardless of whether or not they had reported an episode of concern. Those who had reported tended to attribute their actions to internal factors such as moral strength and a commitment to a professional code. Those who had not or would not report concerns provided accounts which referred to external influences that prevented them from doing so or made reporting pointless. This study provides information about how students account for their actions and omissions in relation to the reporting of poor care. Findings suggest ways educators might increase reporting of concerns.


Induced systemic resistance in Arabidopsis thaliana in response to root inoculation with Pseudomonas fluorescens CHA0.
Root inoculation of Arabidopsis thaliana ecotype Columbia with Pseudomonas fluorescens CHA0r partially protected leaves from the oomycete Peronospora parasitica. The molecular determinants of Pseudomonas fluorescens CHA0r for this induced systemic resistance (ISR) were investigated, using mutants derived from strain CHA0: CHA400 (pyoverdine deficient), CHA805 (exoprotease deficient), CHA77 (HCN deficient), CHA660 (pyoluteorin deficient), CHA631 (2,4-diacetylphloroglucinol [DAPG] deficient), and CHA89 (HCN, DAPG- and pyoluteorin deficient). Only mutations interfering with DAPG production led to a significant decrease in ISR to Peronospora parasitica. Thus, DAPG production in Pseudomonas fluorescens is required for the induction of ISR to Peronospora parasitica. DAPG is known for its antibiotic activity; however, our data indicate that one action of DAPG could be due to an effect on the physiology of the plant. DAPG at 10 to 100 microM applied to roots of Arabidopsis mimicked the ISR effect. CHA0r-mediated ISR was also tested in various Arabidopsis mutants and transgenic plants: NahG (transgenic line degrading salicylic acid [SA]), sid2-1 (nonproducing SA), npr1-1 (non-expressing NPR1 protein), jar1-1 (insensitive to jasmonic acid and methyl jasmonic acid), ein2-1 (insensitive to ethylene), etr1-1 (insensitive to ethylene), eir1-1 (insensitive to ethylene in roots), and pad2-1 (phytoalexin deficient). Only jar1-1, eir1-1, and npr1-1 mutants were unable to undergo ISR. Sensitivity to jasmonic acid and functional NPR1 and EIR1 proteins were required for full expression of CHA0r-mediated ISR. The requirements for ISR observed in this study in Peronospora parasitica induced by Pseudomonas fluorescens CHA0r only partially overlap with those published so far for Peronospora parasitica, indicating a great degree of flexibility in the molecular processes leading to ISR.


Nucleotide divergence vs. gene expression differentiation: comparative transcriptome sequencing in natural isolates from the carrion crow and its hybrid zone with the hooded crow.
Recent advances in sequencing technology promise to provide new strategies for studying population differentiation and speciation phenomena in their earliest phases. We focus here on the black carrion crow (Corvus [corone] corone), which forms a zone of hybridization and overlap with the grey coated hooded crow (Corvus [corone] cornix). However, although these semispecies are taxonomically distinct, previous analyses based on several types of genetic markers did not reveal significant molecular differentiation between them. We here corroborate this result with sequence data obtained from a set of 25 nuclear intronic loci. Thus, the system represents a case of a very early phase of species divergence that requires new molecular approaches for its description. We have therefore generated RNAseq expression profiles using barcoded massively parallel pyrosequencing of brain mRNA from six individuals of the carrion crow and five individuals from a hybrid zone with the hooded crow. We obtained 856 675 reads from two runs, with average read length of 270 nt and coverage of 8.44. Reads were assembled de novo into 19 552 contigs, 70% of which could be assigned to annotated genes in chicken and zebra finch. This resulted in a total of 7637 orthologous genes and a core set of 1301 genes that could be compared across all individuals. We find a clear clustering of expression profiles for the pure carrion crow animals and disperse profiles for the animals from the hybrid zone. These results suggest that gene expression differences may indeed be a sensitive indicator of initial species divergence.


International development in self-directed care.
Self-directed care is an alternative way of delivering services that seeks to empower participants by expanding their degree of choice and control in selecting services. Over the last decade, it has been widely adopted internationally in home and community-based long-term care for people with physical and cognitive disabilities and for seniors. It has been shown to improve satisfaction with services, improve quality of life, and reduce costs compared with services from an agency. A small number of pilot programs are now experimenting with self-directed care in other areas; for example in the management of serious mental illness and other chronic conditions. If positive findings from long-term care can be replicated, self-directed care can make an important contribution to improving health care quality and effectiveness. This issue brief examines a range of innovative self-directed care programs in England, Germany, the Netherlands, and the United States.


Skeletal phenotype amelioration in mucopolysaccharidosis VI requires intervention at the earliest stages of postnatal development.
Mucopolysaccharidosis VI (MPS VI) is a rare lysosomal disease arising from impaired function of the enzyme arylsulfatase B (ARSB). This impairment causes aberrant accumulation of dermatan sulfate, a glycosaminoglycan (GAG) abundant in cartilage. While clinical severity varies along with age at first symptom manifestation, MPS VI usually presents early and strongly affects the skeleton. Current enzyme replacement therapy (ERT) does not provide effective treatment for the skeletal manifestations of MPS VI. This lack of efficacy may be due to an inability of ERT to reach affected cells or to the irreversibility of the disease. To address the question of reversibility of skeletal phenotypes, we generated a conditional by inversion (COIN) mouse model of MPS VI, ArsbCOIN/COIN, wherein Arsb is initially null and can be restored to WT using Cre. We restored Arsb at different times during postnatal development, using a tamoxifen-dependent global Cre driver. By restoring Arsb at P7, P21, and P56-P70, we determined that skeletal phenotypes can be fully rescued if Arsb restoration occurs at P7, while only achieving partial rescue at P21 and no significant rescue at P56-P70. This work has highlighted the importance of early intervention in patients with MPS VI to maximize therapeutic impact.


Optimal Model-free Approach Based on MDL and CHL for Active Brain Identification in fMRI Data Analysis.
Cluster analysis is a robust tool for exploring the underlining structures in data and grouping them with similar objects. In the researches of Functional Magnetic Resonance Imaging (fMRI), clustering approaches attempt to classify voxels depending on their time-course signals into a similar hemodynamic response over time. In this work, a novel unsupervised learning approach is proposed that relies on using Enhanced Neural Gas (ENG) algorithm in fMRI data for comparison with Neural Gas (NG) method, which has yet to be utilized for that aim. The ENG algorithm depends on the network structure of the NG and concentrates on an efficacious prototype-based clustering approach. The comparison outcomes on real auditory fMRI data show that ENG outperforms the NG and statistical parametric mapping (SPM) methods due to its insensitivity to the ordering of input data sequence, various initializations for selecting a set of neurons, and the existence of extreme values (outliers). The findings also prove its capability to discover the exact and real values of a cluster number effectively. Four validation indices are applied to evaluate the performance of the proposed ENG method with fMRI and compare it with a clustering approach (NG algorithm) and model-based data analysis (SPM). These validation indices include the Jaccard Coefficient (JC), Receiver Operating Characteristic (ROC), Minimum Description Length (MDL) value, and Minimum Square Error (MSE). The ENG technique can tackle all shortcomings of NG application with fMRI data, identify the active area of the human brain effectively, and determine the locations of the cluster center based on the MDL value during the process of network learning.


Designing Low-Cost Capacitive-Based Soil Moisture Sensor and Smart Monitoring Unit Operated by Solar Cells for Greenhouse Irrigation Management.
Precise and quick estimates of soil moisture content for the purpose of irrigation scheduling are fundamentally important. They can be accomplished through the continuous monitoring of moisture content in the root zone area, which can be accomplished through automatic soil moisture sensors. Commercial soil moisture sensors are still expensive to be used by famers, particularly in developing countries, such as Egypt. This research aimed to design and calibrate a locally manufactured low-cost soil moisture sensor attached to a smart monitoring unit operated by Solar Photo Voltaic Cells (SPVC). The designed sensor was evaluated on clay textured soils in both lab and controlled greenhouse environments. The calibration results demonstrated a strong correlation between sensor readings and soil volumetric water content (θV). Higher soil moisture content was associated with decreased sensor output voltage with an average determination coefficient (R2) of 0.967 and a root-mean-square error (RMSE) of 0.014. A sensor-to-sensor variability test was performed yielding a 0.045 coefficient of variation. The results obtained from the real conditions demonstrated that the monitoring system for real-time sensing of soil moisture and environmental conditions inside the greenhouse could be a robust, accurate, and cost-effective tool for irrigation management.


Canadian Network for Mood and Anxiety Treatments (CANMAT) 2016 Clinical Guidelines for the Management of Adults with Major Depressive Disorder: Section 2. Psychological Treatments.
The Canadian Network for Mood and Anxiety Treatments (CANMAT) has revised its 2009 guidelines for the management of major depressive disorder (MDD) in adults by updating the evidence and recommendations. The target audiences for these 2016 guidelines are psychiatrists and other mental health professionals. Using the question-answer format, we conducted a systematic literature search focusing on systematic reviews and meta-analyses. Evidence was graded using CANMAT-defined criteria for level of evidence. Recommendations for lines of treatment were based on the quality of evidence and clinical expert consensus. "Psychological Treatments" is the second of six sections of the 2016 guidelines. Evidence-informed responses were developed for 25 questions under 5 broad categories: 1) patient characteristics relevant to using psychological interventions; 2) therapist and health system characteristics associated with optimizing outcomes; 3) descriptions of major psychotherapies and their efficacy; 4) additional psychological interventions, such as peer interventions and computer- and technology-delivered interventions; and 5) combining and/or sequencing psychological and pharmacological interventions. First-line psychological treatment recommendations for acute MDD include cognitive-behavioural therapy (CBT), interpersonal therapy (IPT), and behavioural activation (BA). Second-line recommendations include computer-based and telephone-delivered psychotherapy. Where feasible, combining psychological treatment (CBT or IPT) with antidepressant treatment is recommended because combined treatment is superior to either treatment alone. First-line psychological treatments for maintenance include CBT and mindfulness-based cognitive therapy (MBCT). Patient preference, in combination with evidence-based treatments and clinician/system capacity, will yield the optimal treatment strategies for improving individual outcomes in MDD.


The Pore Loop Domain of TRPV1 Is Required for Its Activation by the Volatile Anesthetics Chloroform and Isoflurane.
The environmental irritant chloroform, a naturally occurring small volatile organohalogen, briefly became the world's most popular volatile general anesthetic (VGA) before being abandoned because of its low therapeutic index. When chloroform comes in contact with skin or is ingested, it causes a painful burning sensation. The molecular basis for the pain associated with chloroform remains unknown. In this study, we assessed the role of transient receptor potential (TRP) channel family members in mediating chloroform activation and the molecular determinants of VGA activation of TRPV1. We identified the subpopulation of dorsal root ganglion (DRG) neurons that are activated by chloroform. Additionally, we transiently expressed wild-type or specifically mutated TRP channels in human embryonic kidney cells and used calcium imaging or whole-cell patch-clamp electrophysiology to assess the effects of chloroform or the VGA isoflurane on TRP channel activation. The results revealed that chloroform activates DRG neurons via TRPV1 activation. Furthermore, chloroform activates TRPV1, and it also activates TRPM8 and functions as a potent inhibitor of the noxious chemical receptor TRPA1. The results also indicate that residues in the outer pore region of TRPV1 previously thought to be required for either proton or heat activation of the channel are also required for activation by chloroform and isoflurane. In addition to identifying the molecular basis of DRG neuron activation by chloroform and the opposing effects chloroform has on different TRP channel family members, the findings of this study provide novel insights into the structural basis for the activation of TRPV1 by VGAs.


Extrachromosomal recombinant adeno-associated virus vector genomes are primarily responsible for stable liver transduction in vivo.
Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocytes in experimental animals. Although the vector genomes are found both as extrachromosomes and as chromosomally integrated forms in hepatocytes, the relative proportion of each has not yet been clearly established. Using an in vivo assay based on the induction of hepatocellular regeneration via a surgical two-thirds partial hepatectomy, we have determined the proportion of integrated and extrachromosomal rAAV genomes in mouse livers and their relative contribution to stable gene expression in vivo. Plasma human coagulation factor IX (hF.IX) levels in mice originating from a chromosomally integrated hF.IX-expressing transposon vector remained unchanged with hepatectomy. This was in sharp contrast to what was observed when a surgical partial hepatectomy was performed in mice 6 weeks to 12 months after portal vein injection of a series of hF.IX-expressing rAAV vectors. At doses of 2.4 x 10(11) to 3.0 x 10(11) vector genomes per mouse (n = 12), hF.IX levels and the average number of stably transduced vector genomes per cell decreased by 92 and 86%, respectively, after hepatectomy. In a separate study, one of three mice injected with a higher dose of rAAV had a higher proportion (67%) of integrated genomes, the significance of which is not known. Nevertheless, in general, these results indicate that, in most cases, no more than approximately 10% of stably transduced genomes integrated into host chromosomes in vivo. Additionally, the results demonstrate that extrachromosomal, not integrated, genomes are the major form of rAAV in the liver and are the primary source of rAAV-mediated gene expression. This small fraction of integrated genomes greatly decreases the potential risk of vector-related insertional mutagenesis associated with all integrating vectors but also raises uncertainties as to whether rAAV-mediated hepatic gene expression can persist lifelong after a single vector administration.


Focal-length measurement by multiple-beam shearing interferometry.
The application of multiple-beam shearing interferometry to lens focal-length measurement is described. A coated shearing plate interferometer was used in transmission to produce sharp multiple-beam fringes that rotate as the collimation of the incoming wave front from the lens under test changes. The test lens was used to collimate light from a point source that was translated longitudinally, and the focal length was determined from the rate of rotation of the fringes as the source moved. This method is simple, accurate, and lends itself to automatic determination of focal length.


Developing measures on the perceptions of the built environment for physical activity: a confirmatory analysis.
Minimal validity evidence exists for scales assessing the built environment for physical activity. The purpose of this study was to assess the test-retest reliability and invariance of a three-factor model (Neighborhood Characteristics, Safety/Crime, and Access to Physical Activity Facilities) across gender, race, geographic location, and level of physical activity. To assess measurement invariance, a random sample of 1,534 adults living in North Carolina or Mississippi completed a computer assisted telephone interview that included items examining perceptions of the neighborhood for physical activity. Construct level test-retest reliability data were collected from a purposeful sample of 106 participants who were administered the questionnaire twice, approximately two weeks apart. Fit indices, Cronbach's alpha, Mokken H and Spearman correlation coefficients (SCC) were used to evaluate configural and co/variance invarianc,e and intraclass correlation coefficients (ICC) were used to assess reliability. Construct test-retest reliability was strong (ICC 0.90 to 0.93). SCC for Neighborhood Characteristics and Crime/Safety were weak with Access (0.21 and 0.25), but strong between Crime/Safety and Neighborhood Characteristics (0.62). Acceptable fit and evidence of measurement invariance was found for gender, race (African American and White), geographic location, and level of physical activity. Fit indices consistently approached or were greater than 0.90 for goodness of fit index, normed fit index, and comparative fit index which is evidence of configural invariance. There was weak support of variance and covariance invariance for all groups that was indicative of factorial validity. Support of the validity and reliability of the three-factor model across groups expands the possibilities for analysis to include latent variable modeling, and suggests these built environment constructs may be used in other settings and populations.


Designing an improved T-cell mobilising CXCL10 mutant through enhanced GAG binding affinity.
The chemokine CXCL10 is released by a plethora of cells, including immune and metastatic cancer cells, following stimulation with interferon-gamma. It acts via its GPC receptor on T-cells attracting them to various target tissues. Glycosaminoglycans (GAGs) are regarded as co-receptors of chemokines, which enable the establishment of a chemotactic gradient for target cell migration. We have engineered human CXCL10 towards improved T-cell mobilisation by implementing a single site-directed mutation N20K into the protein, which leads to a higher GAG binding affinity compared to the wild type. Interestingly, this mutation not only increased T-cell migration in a transendothelial migration assay, the mutant intensified T-cell chemotaxis also in a Boyden chamber set-up thereby indicating a strong role of T-cell-localised GAGs on leukocyte migration. A CXCL10 mutant with increased GAG-binding affinity could therefore potentially serve as a T-cell mobiliser in pathological conditions where the immune surveillance of the target tissue is impaired, as is the case for most solid tumors.


Plasmonic nanograting tip design for high power throughput near-field scanning aperture probe.
We design nanogratings consisting of concentric plasmonic resonance grooves on the metallic sidewalls of near-field scanning probe aperture to increase the power throughput without losing the imaging resolution. Nanograting tip design involves choosing the proper pitch length and the cut location of grooves. Four different nanograting designs are evaluated, as compared with standard single aperture pyramidal near-field scanning probe without grating patterns. We show that, by adding nano-grooves at the location of electromagnetic field intensity-maximum along interface and with the pitch period matching the surface plasmon wavelength, the power throughput can be greatly increased by at least a factor of 530 at 405nm UV wavelength with 100nm diameter aperture probe.


PEG-PLA-Coated and Uncoated Radio-Luminescent CaWO4 Micro- and Nanoparticles for Concomitant Radiation and UV-A/Radio-Enhancement Cancer Treatments.
Currently, there is great interest in the development of ways to achieve the benefits of radiation treatments with reduced negative effects. The present study demonstrates the utilization of radio-luminescent particles (RLPs) as a means to achieve radio-sensitization and enhancement and their ability to affect head- and neck-cancer-cell cultures (in vitro) and xenografts (in vivo). Our approach utilizes a naturally abundant radio-luminescent mineral, calcium tungstate (CaWO4), in its micro or nanoparticulate form for generating secondary UV-A light by γ ray or X-ray photons. In vitro tests demonstrate that unoptimized RLP materials (uncoated CaWO4 (CWO) microparticles (MPs) and PEG-PLA-coated CWO nanoparticles (NPs)) induce a significant enhancement of the tumor-suppressive effect of X-rays and γ rays in both radio-sensitive- and radio-resistant-cancer models; uncoated CWO MPs and PEG-PLA-coated CWO NPs demonstrate comparable radio-sensitization efficacies in vitro. Mechanistic studies reveal that concomitant CaWO4 causes increased mitotic death in radio-resistant cells treated with radiation, whereas CaWO4 sensitizes radio-sensitive cells to X-ray-induced apoptosis and necrosis. The radio-sensitization efficacy of intratumorally injected CaWO4 particles (uncoated CWO MPs and PEG-PLA-coated CWO NPs) is also evaluated in vivo in mouse head- and neck-cancer xenografts. Uncoated CWO MPs suppress tumor growth more effectively than PEG-PLA-coated CWO NPs. On the basis of theoretical considerations, an argument is proposed that uncoated CWO MPs release subtoxic levels of tungstate ions, which cause increased photoelectric-electron-emission effects. The effect of folic acid functionalization on the in vitro radio-sensitization behavior produced by PEG-PLA-coated CWO NPs is studied. Surface folic acid results in a significant improvement in the radio-sensitization efficiency of CaWO4.